New strategies are needed to enhance the efficacy of anti-programmed cell death protein (PD-1) antibody (Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of CQ derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti-PD-1 Ab in melanoma. The combination resulted tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen primed T cells to macrophage conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma specific killing. Genetic or chemical PPT1 inhibition resulted an M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells (MDSCs) in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), tank-binding kinase 1 (TBK1) pathway activation and the secretion of interferon β (IFN-β) in macrophages which was a key component for augmented T cell-mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for a melanoma clinical trial testing this new immunotherapy combination and may also be effective in other cancers.
Gaurav Sharma, Rani Ojha, Estela Noguera-Ortega, Vito W. Rebecca, John Attanasio, Shujing Liu, Shengfu Piao, Jennifer J. Lee, Michael C. Nicastri, Sandra L. Harper, Amruta Ronghe, Vaibhav Jain, Jeffrey D. Winkler, David W. Speicher, Jerome Mastio, Phyllis A Gimotty, Xiaowei Xu, E. John Wherry, Dmitry I. Gabrilovich, Ravi K. Amaravadi
Alpha 1-antitrypsin (AAT) deficiency, a hereditary disorder characterized by low serum levels of functional AAT, is associated with early development of panacinar emphysema. AAT inhibits serine proteases, including neutrophil elastase, protecting the lung from proteolytic destruction. Cigarette smoke, pollution, and inflammatory cell–mediated oxidation of methionine (M) 351 and 358 inactivates AAT, limiting lung protection. In vitro studies using amino acid substitutions demonstrated that replacing M351 with valine (V) and M358 with leucine (L) on a normal M1 alanine (A) 213 background provided maximum antiprotease protection despite oxidant stress. We hypothesized that a onetime administration of a serotype 8 adeno-associated virus (AAV8) gene transfer vector coding for the oxidation-resistant variant AAT (A213/V351/L358; 8/AVL) would maintain antiprotease activity under oxidant stress compared with normal AAT (A213/M351/M358; 8/AMM). 8/AVL was administered via intravenous (IV) and intrapleural (IPL) routes to C57BL/6 mice. High, dose-dependent AAT levels were found in the serum and lung epithelial lining fluid (ELF) of mice administered 8/AVL or 8/AMM by IV or IPL. 8/AVL serum and ELF retained serine protease–inhibitory activity despite oxidant stress while 8/AMM function was abolished. 8/AVL represents a second-generation gene therapy for AAT deficiency providing effective antiprotease protection even with oxidant stress.
Meredith L. Sosulski, Katie M. Stiles, Esther Z. Frenk, Fiona M. Hart, Yuki Matsumura, Bishnu P. De, Stephen M. Kaminsky, Ronald G. Crystal
Platinum-based chemotherapy in combination with immune-checkpoint inhibitors is the current standard of care for patients with advanced lung adenocarcinoma (LUAD). However, tumor progression evolves in most cases. Therefore, predictive biomarkers are needed for better patient stratification and for the identification of new therapeutic strategies, including enhancing the efficacy of chemotoxic agents. Here, we hypothesized that discoidin domain receptor 1 (DDR1) may be both a predictive factor for chemoresistance in patients with LUAD and a potential target positively selected in resistant cells. By using biopsies from patients with LUAD, KRAS-mutant LUAD cell lines, and in vivo genetically engineered KRAS-driven mouse models, we evaluated the role of DDR1 in the context of chemotherapy treatment. We found that DDR1 is upregulated during chemotherapy both in vitro and in vivo. Moreover, analysis of a cohort of patients with LUAD suggested that high DDR1 levels in pretreatment biopsies correlated with poor response to chemotherapy. Additionally, we showed that combining DDR1 inhibition with chemotherapy prompted a synergistic therapeutic effect and enhanced cell death of KRAS-mutant tumors in vivo. Collectively, this study suggests a potential role for DDR1 as both a predictive and prognostic biomarker, potentially improving the chemotherapy response of patients with LUAD.
Marie-Julie Nokin, Elodie Darbo, Camille Travert, Benjamin Drogat, Aurélie Lacouture, Sonia San José, Nuria Cabrera, Béatrice Turcq, Valérie Prouzet-Mauleon, Mattia Falcone, Alberto Villanueva, Haiyun Wang, Michael Herfs, Miguel Mosteiro, Pasi A. Jänne, Jean-Louis Pujol, Antonio Maraver, Mariano Barbacid, Ernest Nadal, David Santamaría, Chiara Ambrogio
One of the most significant adverse post-burn responses is abnormal scar formation, such as keloids. Despite its prolificacy, the underlying pathophysiology of keloid development is unknown. We recently demonstrated that NLRP3 inflammasome, the master regulator of inflammatory and metabolic responses (e.g. aerobic glycolysis), is essential for physiological wound healing. Therefore, burn patients who develop keloids may exhibit altered immunometabolic responses at the site of injury, which interferes with normal healing and portends keloid development. Here, we confirmed keloid NLRP3 activation (caspase-1 (p<0.05), IL1β (p<0.05), IL18 (p<0.01)) and upregulation in Glut1 (p<0.001) and glycolytic enzymes. Burn skin similarly displayed enhanced glycolysis and Glut1 expression (p<0.01). However, Glut1 was significantly higher in keloid compared to non-keloid burn patients (>2 standard deviations above mean). Targeting aberrant glucose metabolism with shikonin, a pyruvate kinase M2 inhibitor, dampened NLRP3-mediated inflammation (caspase-1 (p<0.05), IL1β (p<0.01)) and improved healing in vivo. In summary, burn skin exhibited evidence of Warburg-like metabolism, similar to keloids. Targeting this altered metabolism could change the trajectory towards normal scarring, indicating the clinical possibility of shikonin for abnormal scar prevention.
Roohi Vinaik, Dalia Barayan, Christopher Auger, Abdikarim Abdullahi, Marc G. Jeschke
Tirzepatide (LY3298176) is a dual GIP and GLP-1 receptor agonist under development for the treatment of type 2 diabetes mellitus (T2DM), obesity, and non-alcoholic steatohepatitis. Early phase trials in T2DM indicate that tirzepatide improves clinical outcomes beyond those achieved by a selective GLP-1 receptor agonist. Therefore, we hypothesized that the integrated potency and signaling properties of tirzepatide provide a unique pharmacological profile tailored for improving broad metabolic control. Here, we establish methodology for calculating occupancy of each receptor for clinically efficacious doses of the drug. This analysis reveals a greater degree of engagement of tirzepatide for the GIP receptor (GIPR) than the GLP-1 receptor (GLP-1R), corroborating an imbalanced mechanism of action. Pharmacologically, signaling studies demonstrate that tirzepatide mimics the actions of native GIP at the GIPR but show bias at the GLP-1R to favor cAMP generation over β-arrestin recruitment, coincident with a weaker ability to drive GLP-1R internalization compared with GLP-1. Experiments in primary islets reveal β-arrestin1 limits the insulin response to GLP-1, but not GIP or tirzepatide, suggesting the biased agonism of tirzepatide enhances insulin secretion. Imbalance toward GIPR, combined with distinct signaling properties at the GLP-1R, together may account for the promising efficacy of this new investigational agent.
Francis S. Willard, Jonathan D. Douros, Maria B. N. Gabe, Aaron D. Showalter, David B. Wainscott, Todd M. Suter, Megan E. Capozzi, Wijnand J. C. van der Velden, Cynthia. Stutsman, Guemalli R. Cardona, Shweta Urva, Paul J. Emmerson, Jens J. Holst, David A. D'Alessio, Matthew P. Coghlan, Mette M. Rosenkilde, Jonathan E. Campbell, Kyle W. Sloop
Aromatase inhibitors (AIs) reduce breast cancer recurrence and prolong survival, but up to 30% of patients exhibit recurrence. Using a genome-wide association study of patients entered on MA.27, a phase III randomized trial of anastrozole vs exemestane, we identified a SNP in CUB And Sushi Multiple Domains 1 (CSMD1) associated with breast cancer free interval, with the variant allele associated with fewer distant recurrences. Mechanistically, CSMD1 regulates CYP19 expression in a SNP-, and drug-dependent fashion and this regulation is different among three AIs, anastrozole, exemestane, and letrozole. Overexpression of CSMD1 sensitized AI-resistant cells to anastrozole but not to the other two AIs. The SNP in CSMD1 that was associated with increased CSMD1 and CYP19 expression levels increased anastrozole sensitivity, but not letrozole or exemestane sensitivity. Anastrozole degrades estrogen receptor α (ERα), especially in the presence of estradiol (E2). ER positive breast cancer organoids and AI- or fulvestrant-resistant breast cancer cells were more sensitive to anastrozole plus E2 than to AI alone. Our findings suggest that the CSMD1 SNP might help to predict AI response and anastrozole plus E2 serves as a potential new therapeutic strategy for patients with AI- or fulvestrant-resistant breast cancers.
Junmei Cairns, James N. Ingle, Tanda T. M. Dudenkov, Krishna R. Kalari, Erin E. Carlson, Jie Na, Aman U. Buzdar, Mark E. Robson, Matthew J. Ellis, Paul E. Goss, Lois E. Shepherd, Barbara Goodnature, Matthew P. Goetz, Richard M. Weinshilboum, Hu Li, Mehrab Ghanat Bari, Liewei Wang
Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel tick-borne bunyavirus that recently emerged in East Asian countries. SFTS is characterized by high fever, thrombocytopenia, leukopenia, multiorgan failure, and hemorrhage with case fatality rates of 6.3% to 30%. Neither antivirals nor vaccines are available at present. We previously demonstrated that neutralizing antibodies specific for SFTSV glycoprotein (Gn) played a vital role in the survival of patients with SFTS. Nanobodies from camels present unique properties, such as thermostability, high affinity, and low immunogenicity. In the current study, mammalian expressed SFTSV Gn was used to immunize a camel, and functional nanobodies were isolated from the B cell nanobody library constructed from the immunized animal. Clone SNB02 was selected for in-depth analysis for its inhibition of SFTSV replication both in vitro and in vivo. We showed that SNB02 potently inhibited SFTSV infection and prevented thrombocytopenia in a humanized mouse model and is a potential candidate for therapeutics.
Xilin Wu, Yanlei Li, Bilian Huang, Xiaohua Ma, Linjing Zhu, Nan Zheng, Shijie Xu, Waqas Nawaz, Changping Xu, Zhiwei Wu
De novo lipogenesis (DNL) plays a role in the development of hepatic steatosis. In humans with lipodystrophy, reduced adipose tissue causes lower plasma leptin, insulin resistance, dyslipidemia and ectopic triglyceride (TG) accumulation. We hypothesized that recombinant leptin (metreleptin) for 6 months in 11 patients with lipodystrophy would reduce DNL by decreasing insulin resistance and glycemia, thus reducing circulating and hepatic-TG. The percentage of TG-rich lipoprotein particle (TRLP)-TG derived from DNL (%DNL) was measured by deuterium incorporation from body water into palmitate. At baseline, DNL was elevated with levels similar to levels previously shown in obesity-associated nonalcoholic fatty liver disease (NAFLD). After metreleptin, DNL decreased into the normal range. Similarly, absolute DNL (TRLP-TG x % DNL) decreased by 88% to near-normal levels. Metreleptin improved peripheral insulin sensitivity (hyperinsulinemic-euglycemic clamp) and lowered HbA1c and hepatic-TG. Both before and after metreleptin, DNL positively correlated with insulin resistance, insulin doses, and hepatic-TG, supporting the hypothesis that hyperinsulinemia stimulates DNL and that elevated DNL is integral to the pathogenesis of lipodystrophy-associated NAFLD.These data suggest that leptin-mediated improvement in insulin sensitivity increases clearance of blood glucose by peripheral tissues, reduces hepatic carbohydrate flux, and lowers insulinemia, resulting in DNL reductions, and improvements in hepatic steatosis and dyslipidemia.
Annah P. Baykal, Elizabeth J. Parks, Robert Shamburek, Majid M. Syed-Abdul, Shaji K. Chacko, Elaine Cochran, Megan Startzell, Ahmed M. Gharib, Ronald Ouwerkerk, Khaled Z. Abd-Elmoniem, Peter J. Walter, Mary Walter, Ranganath Muniyappa, Stephanie T. Chung, Rebecca J. Brown
Off-tumor targeting of human antigens is difficult to predict in preclinical animal studies and can lead to serious adverse effects in patients. To address this, we developed a mouse model with stable and tunable human HER2 (hHER2) expression on normal hepatic tissue and compared toxicity between affinity-tuned HER2 CAR T cells (CARTs). In mice with hHER2-high livers, both the high-affinity (HA) and low-affinity (LA) CARTs caused lethal liver damage due to immunotoxicity. Mice with hHER2-low livers, LA-CARTs exhibited less liver damage and lower systemic levels of IFN-γ than HA-CARTs. We then compared affinity-tuned CARTs for their ability to control a hHER2-positive tumor xenograft in our model. Surprisingly, the LA-CARTs outperformed the HA-CARTs with superior antitumor efficacy in vivo. We hypothesized that this was due in part to T cell trafficking differences between LA and HA-CARTs and found that the LA-CARTs migrated out of the liver and infiltrated the tumor sooner than the HA-CARTs. These findings highlight the importance of T cell targeting in reducing toxicity of normal tissue and also in preventing off-tumor sequestration of CARTs, which reduces their therapeutic potency. Our model may be useful to evaluate various CARTs that have conditional expression of more than one scFv.
Mauro Castellarin, Caroline Sands, Tong Da, John Scholler, Kathleen Graham, Elizabeth Buza, Joseph A. Fraietta, Yangbing Zhao, Carl H. June
Musculoskeletal disorders represent the 3rd greatest burden on health in the developed world. Osteoarthritis is the single greatest cause of chronic pain, has no cure, and affects 8.5 and 27 million in the UK and US respectively. Osteoarthritis commonly occurs after joint injury, particularly affecting younger patients. Painful joints are often treated with injections of steroid or hyaluronic acid (HA), but treatments to prevent subsequent joint degeneration remain elusive. In animals, joint injury increases glutamate release into the joint, acting on nerves to cause pain, and joint tissues to cause inflammation and degeneration. This study investigated synovial fluid glutamate concentrations and glutamate receptor (GluR) expression in injured human joints and compared efficacy of GluR antagonists with current treatments in a mouse model of injury-induced osteoarthritis (ACL rupture). GluRs were expressed in ligament and meniscus after knee injury and synovial fluid glutamate concentrations ranged from 19–129 µM. Intra-articular injection of NBQX (GluR antagonist), administered at the time of injury, substantially reduced swelling and degeneration in the mouse ACL rupture model. HA had no effect and depo-medrone reduced swelling for 1 day, but increased degeneration by 50%. Intra-articular administration of NBQX was both symptom and disease modifying to a greater extent than current treatments. There is an opportunity for repurposing related drugs, developed for CNS disorders, with proven safety in man, to prevent injury-induced osteoarthritis. This could quickly reduce the substantial burden associated with osteoarthritis.
Cleo S. Bonnet, Sophie J. Gilbert, Emma J. Blain, Anwen S. Williams, Deborah J. Mason
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